Base Pair Formation Research Articles

Zn2+ ions improve the fidelity of metal-mediated primer extension while suppressing intrinsic and Mn2+-induced mutagenic effects by DNA polymerases.

While Mn2+ ions are well-established for reducing the fidelity of DNA polymerases, leading to the misincorporation of nucleotides, our investigation of the effects of metal ions revealed a contrasting role of Zn2+. Here, we demonstrate that Zn2+ ions enhance the fidelity of DNA polymerases (the 3' → 5' exonuclease-deficient Klenow fragment and Taq DNA polymerase) by suppressing misincorporation during primer extension reactions. Remarkably, Zn2+ ions inhibit both intrinsic misincorporation and Mn2+-induced misincorporation of nucleotides. Furthermore, Zn2+ ions also effectively suppressed misincorporation during metal-mediated primer extension reactions, which involved forming Ag+ and Hg2+ ion-mediated base pairs. These findings suggest that Zn2+ ions inhibit both intrinsic and Mn2+-induced mismatched base pair formation. Consequently, the combined use of Mn2+ and Zn2+ ions may offer a strategy for precisely regulating the fidelity of DNA polymerases. Remarkably, Zn2+ ions even suppress misincorporation in primer extension reactions that rely on metal-mediated base pairs, and conversely, this suggests that DNA polymerases recognize metal-mediated base pairs such as T-Hg2+-T, C-Ag+-A, and C-Ag+-T as relatively stable base pairs. These results imply that Zn2+ ions may also enhance the fidelity of DNA polymerases when incorporating non-canonical nucleobases, potentially paving the way for the expansion of the genetic alphabet.

The effect of the loop on the thermodynamic and kinetic of single base pair in pseudoknot.

RNA pseudoknots are RNA molecules with specialized three-dimensional structures that play important roles in various biological processes. To understand the functions and mechanisms of pseudoknots, it is essential to elucidate their structures and folding pathways. The most fundamental step in RNA folding is the opening and closing of a base pair. The effect of flexible loops on the base pair in pseudoknots remains unclear. In this work, we use molecular dynamics simulations and Markov state model to study the configurations, thermodynamic and kinetic of single base pair in pseudoknots. We find that the presence of the loop leads to a trap state. In addition, the rate-limiting step for the formation of base pair is the disruption of the trap state, rather than the open state to the closed state, which is quite different from the previous studies on non-pseudoknot RNA. For the thermodynamic parameters in pseudoknots, we find that the entropy difference upon opening the base pair between this simulation and the nearest-neighbor model results from the different entropy of different lengths of loop in solution. The thermodynamic parameters of the stack in pseudoknot are close to the nearest-neighbor parameters. The bases on the loop have different distribution patterns in different states, and the slow transition states of the loop are determined by the orientation of the bases.

Structural basis of water-mediated cis Watson-Crick/Hoogsteen base-pair formation in non-CpG methylation.

Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an anti→syntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.

Unveiling the Intermolecular Interactions between Drug 5-Fluorouracil and Watson-Crick/Hoogsteen Base Pairs: A Computational Analysis.

The adsorption of 5-fluorouracil (5FU) on Watson-Crick (WC) base pairs and Hoogsteen (HT) base pairs has been studied using the dispersion-corrected density functional theory (DFT). The adsorption, binding energy, and thermochemistry for the drug 5FU on the WC and HT base pairs were determined. The most stable geometries were near planar geometry, and 5FU has a higher preference for WC than HT base pairs. The adsorption energies of 5FU on nucleobase pairs are consistently higher than pristine nucleobase pairs, indicating that nucleobase pair cleavage is less likely during the adsorption of the 5FU drug. The enthalpy change for the formation of 5FU-DNA base pairs is higher than that for the formation of 5FU-nucleobases and is enthalpy-driven. The E gap of AT base pairs is higher, suggesting that their chemical reactivity toward further reaction would be less than that of GC base pairs. The electron density difference (EDD) analysis shows a significant decrease in electron density in aromatic regions on the purine bases (adenine/guanine) compared to the pyrimidine bases. The MESP diagram of the stable 5FU-nucleobase pair complexes shows a directional interaction, with the positive regions in a molecule interacting with the negative region of other molecules. The atoms in molecule analysis show that the ρ(r) values of C=O···H-N are higher than those of N···H/N-H···O. The N···H intermolecular bonds between the base pair/drug and nucleobases are weak, closed shell interactions and are electrostatic in nature. The noncovalent interaction analysis shows that several new spikes are engendered along with an increase in their strength, which indicates that the H-bonding interactions are stronger and play a dominant role in stabilizing the complexes. Energy decomposition analysis shows that the drug-nucleobase pair complex has a marginal increase in the electrostatic contributions compared to nucleobase pair complexes.

An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.

The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.

Specific binding of Ag+ to central C[sbnd]C mismatched base pair but not terminal C[sbnd]C pair in duplex DNA

Metal ion-nucleic acid interactions are important for their contribution in structure formation and their potential applications in nanotechnology. Hg2+ and Ag+ bind to T–T and CC mismatched base pairs, respectively, at the center of duplex DNA to form T–Hg–T and C–Ag–C. Although primer-extension by DNA polymerases with Hg2+ incorporated thymidine 5′-triphosphate to form T–Hg–T, the same reaction with Ag+ did not incorporate deoxycytidine 5′-triphosphate to form C–Ag–C. Here, isothermal titration calorimetric analyses to examine the effect of CC position in duplex DNA on Ag+ binding demonstrated that Ag+ did not bind to the terminal CC base pair in duplex, but it bound to the central CC base pair in duplex at 1:1 molar ratio with 9 × 105 M–1 binding constant. Ag+ did not bind to the terminal and central C–A, C–G, and C–T base pairs in duplex. These findings are useful for developing efficient metal-mediated base pair formation in nanotechnology.

Base pair dynamics, electrostatics, and thermodynamics at the LTR-III quadruplex:duplex junction

G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.

One-Pot Formation of Pairing Proto-RNA Nucleotides and Their Supramolecular Assemblies.

Most contemporary theories for the chemical origins of life include the prebiotic synthesis of informational polymers, including strong interpretations of the RNA World hypothesis. Existing challenges to the prebiotic emergence of RNA have encouraged exploration of the possibility that RNA was preceded by an ancestral informational polymer, or proto-RNA, that formed more easily on the early Earth. We have proposed that the proto-nucleobases of proto-RNA would have readily formed glycosides with ribose and that these proto-nucleosides would have formed base pairs as monomers in aqueous solution, two properties not exhibited by the extant nucleosides or nucleotides. Here we demonstrate that putative proto-nucleotides of the model proto-nucleobases barbituric acid and melamine can be formed in the same one-pot reaction with ribose-5-phosphate. Additionally, the proto-nucleotides formed in these reactions spontaneously form assemblies that are consistent with the presence of Watson-Crick-like base pairs. Together, these results provide further support for the possibility that heterocycles closely related to the extant bases of RNA facilitated the prebiotic emergence of RNA-like molecules, which were eventually replaced by RNA over the course of chemical and biological evolution.

Sequence Information Transfer in Oligoarylacetylenes

AbstractThe ability of genetic biopolymers to store and transfer information provides the mechanism for the evolution of function from a population of sequences. Synthetic polymers with the ability to transmit information are exceedingly rare. This work demonstrates template‐directed information transfer in oligoarylacetylene trimers that use reversible covalent bonds to form base pairing interactions. Information was encoded as a sequence of aniline and benzaldehyde subunits linked together by a diethynylbenzene backbone. Trimers formed sequence‐specific, imine‐linked monomer‐template complexes, which could be oligomerized using palladium mediated Sonogashira coupling to generate daughter trimers with complementary sequences.

Formation and Effects of Upstream DNA-RNA Base Pairing in Telomerase.

Telomere elongation by telomerase consists of two types of translocation: duplex translocation during each repeat synthesis and template translocation at the end of repeat synthesis. Our replica exchange molecular dynamics simulations show that in addition to the Watson-Crick interactions in the active site, templating RNA can also form base pairs with the upstream regions of DNA, mostly with the second upstream DNA repeat with respect to the 3'-end. At the end of the repeat synthesis, dG10-P442 and dG11-N446 hydrogen bonds form. Then, active-site base pairs dissociate one by one, and the RNA bases reanneal with the complementary base on the upstream DNA repeat. For each dissociating base pair a new one forms, thus conserving the number of base pairs during template translocation.

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

The 3' end of the coding region of senecavirus A contains a highly conserved sequence that potentially forms a stem-loop structure required for virus rescue.

Senecavirus A (SVA) can cause a vesicular disease in swine. It is a positive-strand RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Positive-strand RNA viruses possess positive-sense, single-stranded genomes whose untranslated regions (UTRs) have been reported to contain cis-acting RNA elements. In the present study, a total of 100 SVA isolates were comparatively analyzed at the genome level. A highly conserved fragment (HCF) was found to be located in the 3D sequence and to be close to the 3' UTR. The HCF was computationally predicted to form a stem-loop structure. Eight synonymous mutations can individually disrupt the formation of a single base pair within the stem region. We found that SVA itself was able to tolerate each of these mutations alone, as evidenced by the ability to rescue all eight single-site mutants from their individual cDNA clones, and all of them were genetically stable during serial passaging. However, the replication-competent SVA could not be rescued from another cDNA clone containing all eight mutations. The failure to recover SVA might be attributed to disruption of the predicted stem-loop structure, whereas introduction of a wild-type HCF into the cDNA clone with eight mutations still had no effect on virus recovery. These results suggest that the putative stem-loop structure at the 3' end of the 3D sequence is a cis-acting RNA element that is required for SVA growth.

Photoinduced electron transfer detection method for identifying UGT1A1*28 microsatellites.

During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.

RNA covariation at helix-level resolution for the identification of evolutionarily conserved RNA structure.

Many biologically important RNAs fold into specific 3D structures conserved through evolution. Knowing when an RNA sequence includes a conserved RNA structure that could lead to new biology is not trivial and depends on clues left behind by conservation in the form of covariation and variation. For that purpose, the R-scape statistical test was created to identify from alignments of RNA sequences, the base pairs that significantly covary above phylogenetic expectation. R-scape treats base pairs as independent units. However, RNA base pairs do not occur in isolation. The Watson-Crick (WC) base pairs stack together forming helices that constitute the scaffold that facilitates the formation of the non-WC base pairs, and ultimately the complete 3D structure. The helix-forming WC base pairs carry most of the covariation signal in an RNA structure. Here, I introduce a new measure of statistically significant covariation at helix-level by aggregation of the covariation significance and covariation power calculated at base-pair-level resolution. Performance benchmarks show that helix-level aggregated covariation increases sensitivity in the detection of evolutionarily conserved RNA structure without sacrificing specificity. This additional helix-level sensitivity reveals an artifact that results from using covariation to build an alignment for a hypothetical structure and then testing the alignment for whether its covariation significantly supports the structure. Helix-level reanalysis of the evolutionary evidence for a selection of long non-coding RNAs (lncRNAs) reinforces the evidence against these lncRNAs having a conserved secondary structure.

Hydrogen-Bonding Interactions of 8-Substituted Purine Derivatives.

Hydrogen bonding between nucleobases is a crucial noncovalent interaction for life on Earth. Canonical nucleobases form base pairs according to two main geometries: Watson-Crick pairing, which enables the static functions of nucleic acids, such as the storing of genetic information; and Hoogsteen pairing, which facilitates the dynamic functions of these biomacromolecules. This precisely tuned system can be affected by oxidation or substitution of nucleobases, leading to changes in their hydrogen-bonding patterns. This paper presents an investigation into the intermolecular interactions of various 8-substituted purine derivatives with their hydrogen-bonding partners. The systems were analyzed using nuclear magnetic resonance spectroscopy and density functional theory calculations. Our results demonstrate that the stability of hydrogen-bonded complexes, or base pairs, depends primarily on the number of intermolecular H-bonds and their donor-acceptor alternation. No strong preferences for a particular geometry, either Watson-Crick or Hoogsteen, were found.

Scaling properties of RNA as a randomly branching polymer.

Formation of base pairs between the nucleotides of a ribonucleic acid (RNA) sequence gives rise to a complex and often highly branched RNA structure. While numerous studies have demonstrated the functional importance of the high degree of RNA branching-for instance, for its spatial compactness or interaction with other biological macromolecules-RNA branching topology remains largely unexplored. Here, we use the theory of randomly branching polymers to explore the scaling properties of RNAs by mapping their secondary structures onto planar tree graphs. Focusing on random RNA sequences of varying lengths, we determine the two scaling exponents related to their topology of branching. Our results indicate that ensembles of RNA secondary structures are characterized by annealed random branching and scale similarly to self-avoiding trees in three dimensions. We further show that the obtained scaling exponents are robust upon changes in nucleotide composition, tree topology, and folding energy parameters. Finally, in order to apply the theory of branching polymers to biological RNAs, whose length cannot be arbitrarily varied, we demonstrate how both scaling exponents can be obtained from distributions of the related topological quantities of individual RNA molecules with fixed length. In this way, we establish a framework to study the branching properties of RNA and compare them to other known classes of branched polymers. By understanding the scaling properties of RNA related to its branching structure, we aim to improve our understanding of the underlying principles and open up the possibility to design RNA sequences with desired topological properties.

Predicting DNA kinetics with a truncated continuous-time Markov chain method

Predicting the kinetics of reactions involving nucleic acid strands is a fundamental task in biology and biotechnology. Reaction kinetics can be modeled as an elementary step continuous-time Markov chain, where states correspond to secondary structures and transitions correspond to base pair formation and breakage. Since the number of states in the Markov chain could be large, rates are determined by estimating the mean first passage time from sampled trajectories. As a result, the cost of kinetic predictions becomes prohibitively expensive for rare events with extremely long trajectories. Also problematic are scenarios where multiple predictions are needed for the same reaction, e.g., under different environmental conditions, or when calibrating model parameters, because a new set of trajectories is needed multiple times. We propose a new method, called pathway elaboration, to handle these scenarios. Pathway elaboration builds a truncated continuous-time Markov chain through both biased and unbiased sampling. The resulting Markov chain has moderate state space size, so matrix methods can efficiently compute reaction rates, even for rare events. Also the transition rates of the truncated Markov chain can easily be adapted when model or environmental parameters are perturbed, making model calibration feasible. We illustrate the utility of pathway elaboration on toehold-mediated strand displacement reactions, show that it well-approximates trajectory-based predictions of unbiased elementary step models on a wide range of reaction types for which such predictions are feasible, and demonstrate that it performs better than alternative truncation-based approaches that are applicable for mean first passage time estimation. Finally, in a small study, we use pathway elaboration to optimize the Metropolis kinetic model of Multistrand, an elementary step simulator, showing that the optimized parameters greatly improve reaction rate predictions. Our framework and dataset are available at https://github.com/DNA-and-Natural-Algorithms-Group/PathwayElaboration.

Flanking strand separation activity of RecA nucleoprotein filaments in DNA strand exchange reactions.

The recombinase RecA/Rad51 ATPase family proteins catalyze paramount DNA strand exchange reactions that are critically involved in maintaining genome integrity. However, it remains unclear how DNA strand exchange proceeds when encountering RecA-free defects in recombinase nucleoprotein filaments. Herein, by designing a series of unique substrates (e.g. truncated or conjugated incoming single-stranded DNA, and extended donor double-stranded DNA) and developing a two-color alternating excitation-modified single-molecule real-time fluorescence imaging assay, we resolve the two key steps (donor strand separation and new base-pair formation) that are usually inseparable during the reaction, revealing a novel long-range flanking strand separation activity of synaptic RecA nucleoprotein filaments. We further evaluate the kinetics and free energetics of strand exchange reactions mediated by various substrates, and elucidate the mechanism of flanking strand separation. Based on these findings, we propose a potential fundamental molecular model involved in flanking strand separation, which provides new insights into strand exchange mechanism and homologous recombination.

Mechanism of ligand binding signal transduction in a transcriptional riboswitch.

Riboswitches are noncoding RNA molecules that regulate gene expression in bacteria. The riboswitch's aptamer domain (AD) senses the cognate ligand and induces a structural change in the expression platform (EP) to signal on/off to the gene expression machinery. Despite extensive studies, the question of how the ligand binding signal is transmitted from the AD to EP resulting in the structural transitions in the EP remains unanswered. We studied the ligand binding signal transduction from AD to EP in the preQ1-sensing transcriptional riboswitch. In the absence of preQ1, both AD and EP individually form stem-loops with no structural overlap between the two stem-loops. The segment between the AD and EP stem loops is rich in adenine. In the presence of preQ1, AD forms a pseudoknot to encapsulate preQ1. We performed coarse-grained molecular dynamics simulations of full-length preQ1 riboswitch in the presence of Mg2+ and preQ1 ligand. In the absence of preQ1 and variation in Mg2+ concentration ([Mg2+]), we observed that AD populates only stem-loop and does not form the pseudoknot required to capture the ligand. Only in the presence of preQ1 AD attains the pseudoknot conformation by wrapping the A-rich region around the pre-existing stem-loop to facilitate ligand binding. The pseudoknot formation at AD allows EP to form the terminator helix and sequester the poly-U through strand invasion. The wrapping of the A-rich region around the AD stem-loop forces the stem-loop at EP to rupture and slide by 10 nucleotides towards 3'-end and form the terminator helix. The propensity of pseudoknot and terminator formation through strand invasion in the presence of preQ1 is enhanced with increment in [Mg2+]. We show that the competition between base-pair formation and helix sliding rate is crucial for the ligand binding signal transduction.

Supramolecular interactions in salts/cocrystals involving pyrimidine derivatives of sulfonate/carboxylic acid.

The crystal structures of three compounds involving aminopyrimidine derivatives are reported, namely, 5-fluorocytosinium sulfanilate-5-fluorocytosine-4-azaniumylbenzene-1-sulfonate (1/1/1), C4H5FN3O+·C6H6NO3S-·C4H4FN3O·C6H7NO3S, I, 5-fluorocytosine-indole-3-propionic acid (1/1), C4H4FN3O·C11H11NO2, II, and 2,4,6-triaminopyrimidinium 3-nitrobenzoate, C4H8N5+·C7H4NO4-, III, which have been synthesized and characterized by single-crystal X-ray diffraction. In I, there are two 5-fluorocytosine (5FC) molecules (5FC-A and 5FC-B) in the asymmetric unit, with one of the protons disordered between them. 5FC-A and 5FC-B are linked by triple hydrogen bonds, generating two fused rings [two R22(8) ring motifs]. The 5FC-A molecules form a self-complementary base pair [R22(8) ring motif] via a pair of N-H...O hydrogen bonds and the 5FC-B molecules form a similar complementary base pair [R22(8) ring motif]. The combination of these two types of pairing generates a supramolecular ribbon. The 5FC molecules are further hydrogen bonded to the sulfanilate anions and sulfanilic acid molecules via N-H...O hydrogen bonds, generating R44(22) and R66(36) ring motifs. In cocrystal II, two types of base pairs (homosynthons) are observed via a pair of N-H...O/N-H...N hydrogen bonds, generating R22(8) ring motifs. The first type of base pair is formed by the interaction of an N-H group and the carbonyl O atom of 5FC molecules through a couple of N-H...O hydrogen bonds. Another type of base pair is formed via the amino group and a pyrimidine ring N atom of the 5FC molecules through a pair of N-H...N hydrogen bonds. The base pairs (via N-H...N hydrogen bonds) are further bridged by the carboxyl OH group of indole-3-propionic acid and the O atom of 5FC through O-H...O hydrogen bonds on either side of the R22(8) motif. This leads to a DDAA array. In salt III, one of the N atoms of the pyrimidine ring is protonated and interacts with the carboxylate group of the anion through N-H...O hydrogen bonds, leading to the primary ring motif R22(8). Furthermore, the 2,4,6-triaminopyrimidinium (TAP) cations form base pairs [R22(8) homosynthon] via N-H...N hydrogen bonds. A carboxylate O atom of the 3-nitrobenzoate anion bridges two of the amino groups on either side of the paired TAP cations to form another ring [R32(8)]. This leads to the generation of a quadruple DADA array. The crystal structures are further stabilized by π-π stacking (I and III), C-H...π (I and II), C-F...π (I) and C-O...π (II) interactions.